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Interactions between hepatocytes and liver sinusoidal endothelial cells (LSECs) are essential for the development and maintenance of hepatic phenotypic functions. We report the assembly of three-dimensional liver sinusoidal mimics comprised of primary rat hepatocytes, LSECs, and an intermediate chitosan–hyaluronic acid polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30 to 55 nm and exhibited a shear modulus of ∼100 kPa. Hepatocyte–PEM cellular constructs exhibited stable urea and albumin production over a 7-day period, and these values were either higher or similar to cells cultured in a collagen sandwich. This is of significance because the thickness of a collagen gel is ∼1000-fold higher than the height of the chitosan–hyaluronic acid PEM. In the hepatocyte–PEM–LSEC liver-mimetic cellular constructs, LSEC phenotype was maintained, and these cultures exhibited stable urea and albumin production. CYP1A1/2 activity measured over a 7-day period was significantly higher in the hepatocyte–PEM–LSEC constructs than in collagen sandwich cultures. A 16-fold increase in CYP1A1/2 activity was observed for hepatocyte–PEM–10,000 LSEC samples, thereby suggesting that interactions between hepatocytes and LSECs are critical in enhancing the detoxification capability in hepatic cultures in vitro.

The ability of the liver to regenerate is crucial to protect liver function after injury and during chronic disease. Increases in hepatocyte growth factor (HGF) in liver sinusoidal endothelial cells (LSECs) are thought to drive liver regeneration. However, in contrast to endothelial progenitor cells, mature LSECs express little HGF. Therefore, we sought to establish in rats whether liver injury causes BM LSEC progenitor cells to engraft in the liver and provide increased levels of HGF and to examine the relative contribution of resident and BM LSEC progenitors. LSEC label-retaining cells and progenitors were identified in liver and LSEC progenitors in BM. BM LSEC progenitors did not contribute to normal LSEC turnover in the liver. However, after partial hepatectomy, BM LSEC progenitor proliferation and mobilization to the circulation doubled. In the liver, one-quarter of the LSECs were BM derived, and BM LSEC progenitors differentiated into fenestrated LSECs. When irradiated rats underwent partial hepatectomy, liver regeneration was compromised, but infusion of LSEC progenitors rescued the defect. Further analysis revealed that BM LSEC progenitors expressed substantially more HGF and were more proliferative than resident LSEC progenitors after partial hepatectomy. Resident LSEC progenitors within their niche may play a smaller role in recovery from partial hepatectomy than BM LSEC progenitors, but, when infused after injury, these progenitors engrafted and expanded markedly over a 2-month period. In conclusion, LSEC progenitor cells are present in liver and BM, and recruitment of BM LSEC progenitors is necessary for normal liver regeneration.

Liver Sinusoidal Endothelial Cells (LSEC) differ, both structurally and functionally, from endothelial cells (EC) lining blood vessels of other tissues. For example, in contrast to other EC, LSEC posses fenestrations, have low detectable levels of PECAM-1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE-1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli, namely, extracellular matrix (ECM) and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte-fibroblast) we were able to prolong the expression of both SE-1 and phenotypic functions of LSEC such as Factor VIII activity in co-cultured LSECs through the production of short-range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE-1 as CD32b. Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease.

The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease.

Background

Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered in the hepatic sinusoids is in general considerably lower than the oxygen tension in the air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions with regard to oxygen would expect to improve cell survival, structure and function. In this study LSECs were isolated from rats and cultured under either 5% (normoxic) or 20% (hyperoxic) oxygen tensions, and several morpho-functional features were compared.

Results

Cultivation of LSECs under normoxia, as opposed to hyperoxia improved the survival of LSECs and scavenger receptor-mediated endocytic activity, reduced the production of the pro-inflammatory mediator, interleukin-6 and increased the production of the anti-inflammatory cytokine, interleukin-10. On the other hand, fenestration, a characteristic feature of LSECs disappeared gradually at the same rate regardless of the oxygen tension. Expression of the cell-adhesion molecule, ICAM-1 at the cell surface was slightly more elevated in cells maintained at hyperoxia. Under normoxia, endogenous generation of hydrogen peroxide was drastically reduced whereas the production of nitric oxide was unaltered. Culture decline in high oxygen-treated cultures was abrogated by administration of catalase, indicating that the toxic effects observed in high oxygen environments is largely caused by endogenous production of hydrogen peroxide.

Conclusion

Viability, structure and many of the essential functional characteristics of isolated LSECs are clearly better preserved when the cultures are maintained under more physiologic oxygen levels. Endogenous production of hydrogen peroxide is to a large extent responsible for the toxic effects observed in high oxygen environments.

Background

The liver may have a role in peripheral tolerance, by serving as a site for trapping, apoptosis and phagocytosis of activated T cells. It is not known which hepatic cells are involved in these processes. It was hypothesised that liver sinusoidal endothelial cells (LSEC) which are strategically placed for participation in the regulation of sinusoidal blood flow, and express markers involved in recognition, sequestration and apoptosis, may contribute to peripheral tolerance by inducing apoptosis of activated T cells.

Methods

By using immunoassays and western blot analysis, the fate of activated T cells when incubated with human LSEC isolated from normal healthy livers was investigated.

Results

Evidence that activated (approximately 30%) but not non‐activated T cells undergo apoptosis on incubation with human LSEC in mixed cell cultures is provided. No difference in the results was observed when unstimulated and cytokine‐stimulated LSEC were used. T cell–LSEC contact is required for induction of apoptosis. Apoptosis induced by LSEC was associated with caspase 8 and 3 activity and strong expression of the proapoptotic molecule Bak. Transforming growth factor β (TGFβ) produced constitutively by LSEC is partly responsible for the caspase‐induced apoptosis, as neutralising antibodies to TGFβ markedly attenuated apoptosis, up regulated the antiapoptotic molecule Bcl‐2 and partially blocked caspase‐3 activity.

Conclusion

These findings broaden the potential role of LSEC in immune tolerance and homeostasis of the immune system. This study may provide insight for exploring the mechanisms of immune tolerance by liver allografts, immune escape by some liver pathogens including hepatitis C and pathogenesis of liver diseases.

We evaluated the kinetics by which rat liver sinusoidal endothelial cells (LSECs) are repopulated in the reperfused transplanted liver after 18 hours of cold ischemic storage. We found that the majority of LSECs in livers cold-stored for 18 hours in University of Wisconsin solution are seriously compromised and often are retracted before transplantation. Sinusoids rapidly re-endothelialize within 48 hours of transplantation, and repopulation is coincident with up-regulation of hepatocyte vascular endothelial growth factor expression and vascular endothelial growth factor receptor-2 expression on large vessel endothelial cells and repopulating LSECs. Although re-endothelialization occurs rapidly, we show here, using several high-resolution imaging techniques and 2 different rat liver transplantation models, that engraftment of bone marrow–derived cells into functioning LSECs is routinely between 1% and 5%.

Conclusion

Bone marrow plays a measurable but surprisingly limited role in the rapid repopulation and functional engraftment of bone marrow–derived LSECs after cold ischemia and warm reperfusion.

Background

The liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC.

Results

Endocytosis of five 125I-labelled molecules: collagen α-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of α-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells.

Conclusion

Pig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC.

Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage.

AIM: To investigate whether irradiation (IR) and partial hepatectomy (PH) may prepare the host liver for non-parenchymal cell (NPC) transplantation.

METHODS: Livers of dipeptidyl peptidase IV (DPPIV)-deficient rats were pre-conditioned with external beam IR (25 Gy) delivered to two-thirds of the right liver lobules followed by a one-third PH of the untreated lobule. DPPIV-positive liver cells (NPC preparations enriched for liver sinusoidal endothelial cells (LSECs) and hepatocytes) were transplanted via the spleen into the recipient livers. The extent and quality of donor cell engraftment and growth was studied over a long-term interval of 16 wk after transplantation.

RESULTS: Host liver staining demonstrated 3 different repopulation types. Well defined clusters of donor-derived hepatocytes with canalicular expression of DPPIV were detectable either adjacent to or in between large areas of donor cells (covering up to 90% of the section plane) co-expressing the endothelial marker platelet endothelial cell adhesion molecule. The third type consisted of formations of DPPIV-positive duct-like structures which co-localized with biliary epithelial CD49f.

CONCLUSION: Liver IR and PH as a preconditioning stimulus enables multiple cell liver repopulation by donor hepatocytes, LSECs, and bile duct cells.

Liver sinusoidal endothelial cells (LSECs) play an essential role in systemic waste clearance by effective endocytosis of blood-borne waste macromolecules. We aimed to study LSECs’ scavenger function during aging, and whether age-related morphological changes (eg, defenestration) affect this function, in F344/BN F1 rats. Endocytosis of the scavenger receptor ligand formaldehyde-treated serum albumin was significantly reduced in LSECs from old rats. Ligand degradation, LSEC protein expression of the major scavenger receptors for formaldehyde-treated serum albumin endocytosis, stabilin-1 and stabilin-2, and their staining patterns along liver sinusoids, was similar at young and old age, suggesting that other parts of the endocytic machinery are affected by aging. Formaldehyde-treated serum albumin uptake per cell, and cell porosity evaluated by electron microscopy, was not correlated, indicating that LSEC defenestration is not linked to impaired endocytosis. We report a significantly reduced LSEC endocytic capacity at old age, which may be especially important in situations with increased circulatory waste loads.

Tissue homeostasis and remodeling are processes that involve high turnover of biological macromolecules. Many of the waste molecules that are by-products or degradation intermediates of biological macromolecule turnover enter the circulation and are subsequently cleared by liver sinusoidal endothelial cells (LSEC). Besides the mannose receptor, stabilin-1 and stabilin-2 are the major scavenger receptors expressed by LSEC. To more clearly elucidate the functions of stabilin-1 and -2, we have generated mice lacking stabilin-1, stabilin-2, or both stabilin-1 and -2 (Stab1–/–Stab2–/– mice). Mice lacking either stabilin-1 or stabilin-2 were phenotypically normal; however, Stab1–/–Stab2–/– mice exhibited premature mortality and developed severe glomerular fibrosis, while the liver showed only mild perisinusoidal fibrosis without dysfunction. Upon kidney transplantation into WT mice, progression of glomerular fibrosis was halted, indicating the presence of profibrotic factors in the circulation of Stab1–/–Stab2–/– mice. While plasma levels of known profibrotic cytokines were unaltered, clearance of the TGF-β family member growth differentiation factor 15 (GDF-15) was markedly impaired in Stab1–/–Stab2–/– mice but not in either Stab1–/– or Stab2–/– mice, indicating that it is a common ligand of both stabilin-1 and stabilin-2. These data lead us to conclude that stabilin-1 and -2 together guarantee proper hepatic clearance of potentially noxious agents in the blood and maintain tissue homeostasis not only in the liver but also distant organs.

The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.

Author Summary

The liver has long been known as the garbage dump of the body, capable of rapidly removing hazardous pathogens and useless particles from the blood stream, thereby protecting the host. The only cell doing the removal has been thought to be the liver's macrophages. This is likely true for larger particles such as bacteria. But for smaller particles the size of virus or small antibody-antigen complexes, macrophages are probably not the cell responsible for the bulk of removal. We suggest, rather, it is the endothelial cell of the liver's blood circulatory system that takes up and destroys the majority of virus, doing so quickly (minutes) and extensively (>90%), leaving only a small fraction of circulating virus to infect the body in ways peculiar to each virus. To test this possibility, we infused mice intravenously with a harmless common cold virus and tracked its destination by molecular and microscopy methods. Affirming our conjecture, we found that ∼90% of the infused virus homed to the endothelium of the liver and ∼10% went to its macrophages. These data support a unique role, generally underappreciated, for the liver endothelium in viral clearance.

Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.

A critical hepatic function is the maintenance of optimal bile acid (BA) compositions to achieve cholesterol homeostasis. BAs are rarely quantified to assess hepatic phenotype in vitro since existing analytical techniques have inadequate resolution. We report a detailed investigation into the biosynthesis and homeostasis of eight primary rat BAs in conventional in vitro hepatocyte cultures and in an engineered liver mimic. The three-dimensional (3D) liver mimic was assembled with layers of primary rat hepatocytes and liver sinusoidal endothelial cells. A high-pressure liquid chromatography and mass spectrometry technique was developed with a detection limit of 1 ng/mL for each BA, which is significantly lower than previous approaches. Over a 2-week culture, only 3D liver mimics exhibited the ratio of conjugated cholic acid to chenodeoxycholic acid that has been observed in vivo. This ratio, an important marker of BA homeostasis, was significantly higher in stable collagen sandwich cultures indicating significant deviation from physiological behavior. The biosynthesis of tauro-β-muricholic acid, a key primary rat BA, doubled only in the engineered liver mimics while decreasing in the other systems. These trends demonstrate that the 3D liver mimics provide a unique platform to study hepatic metabolism.

Background

Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from (GalT)-deficient and wild pigs.

Methods

Isolated LSEC from wildtype and GalT pigs were expose to human and baboon sera, IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent Assay. Levels of complement activated cytotoxicity (CAC) were also determined by a fluorescent Live Dead Assay and by the quantification of LDH release.

Results

IgM binding to GalT KO LSECs was significantly lower (80% human and 87% baboon) compare to wildtype pig LSEC. IgG binding was low all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wildtype LSECs and LDH release levels were reduced by 37% and 57% respectively.

Conclusions

LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compare to LSECs from wild pigs when exposed to human serum. Though insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

Background

Numerous studies in rats and a few other mammalian species, including man, have shown that the sinusoidal cells constitute an important part of liver function. In the pig, however, which is frequently used in studies on liver transplantation and liver failure models, our knowledge about the function of hepatic sinusoidal cells is scarce. We have explored the scavenger function of pig liver sinusoidal endothelial cells (LSEC), a cell type that in other mammals performs vital elimination of an array of waste macromolecules from the circulation.

Results

125I-macromolecules known to be cleared in the rat via the scavenger and mannose receptors were rapidly removed from the pig circulation, 50% of the injected dose being removed within the first 2–5 min following injection. Fluorescently labeled microbeads (2 μm in diameter) used to probe phagocytosis accumulated in Kupffer cells only, whereas fluorescently labeled soluble macromolecular ligands for the mannose and scavenger receptors were sequestered only by LSEC. Desmin-positive stellate cells accumulated no probes. Isolation of liver cells using collagenase perfusion through the portal vein, followed by various centrifugation protocols to separate the different liver cell populations yielded 280 × 107 (range 50–890 × 107) sinusoidal cells per liver (weight of liver 237.1 g (sd 43.6)). Use of specific anti-Kupffer cell- and anti-desmin antibodies, combined with endocytosis of fluorescently labeled macromolecular soluble ligands indicated that the LSEC fraction contained 62 × 107 (sd 12 × 107) purified LSEC. Cultured LSEC avidly endocytosed ligands for the mannose and scavenger receptors.

Conclusions

We show here for the first time that pig LSEC, similar to what has been found earlier in rat LSEC, represent an effective scavenger system for removal of macromolecular waste products from the circulation.

Improvements in the treatment of primary biliary cirrhosis may depend upon dissection of mechanisms that determine recruitment of mononuclear cells to intralobular bile ducts, including the role of the chemokine-adhesion molecule CX3CL1 (fractalkine). We submit that there are unique interactions between intrahepatic biliary epithelial cells (BEC), endothelial cells (EC), liver sinusoidal endothelial cells (LSEC) and liver infiltrating mononuclear cells (LMC), and that such interactions will in part dictate the biliary-specific inflammatory response. To address this, we studied fresh explanted liver from pre-transplant patients with PBC and with inflammatory liver disease due to viral infection, designated as ‘disease controls’, and biopsy material from patients with a discrete liver tumor, ‘normal controls’. Using this clinical material, we isolated and stimulated BEC, EC, LSEC, and LMC with a panel of toll-like receptor (TLR) ligands. We also studied the interactions of these cell populations with LMC with respect to adhesion capability and production of TNF-α. Finally, we used fresh biopsy samples to evaluate mononuclear cells around intrahepatic biliary ductules using mAb specific to CD68 or CD154, markers for monocytes/macrophages and activated T cells respectively. We report herein that there are common properties of EC, LSEC and BEC, whether derived from PBC or viral hepatitis, but there are also significant differences, particularly in the potential in PBC for LMC to adhere to EC and BEC, and to produce TNF-α; such properties were associated with augmented CX3CL1 production by BEC from PBC liver. The processes defined herein suggest potential novel biotherapies for biliary specific inflammation.

It is increasingly appreciated that since cell and tissue functions are regulated by chemomechanical stimuli, precise control over such stimuli will improve the functionality of tissue models. However, due to the inherent difficulty in decoupling these cues as presented by extracellular materials, few studies have explored the independent modulation of biochemical and mechanical stimuli towards the manipulation of sustained cellular processes. Here, we demonstrate that both mechanical compliance and ligand presentation of synthetic, weak polyelectrolyte multilayers (PEMs) can be tuned independently to influence the adhesion and liver-specific functions of primary rat hepatocytes over extended in vitro culture (two weeks). These synthetic PEMs exhibited elastic moduli E ranging over 200 kPa -< E < 142 MPa, as much as one thousand-fold more compliant than tissue-culture polystyrene (E ∼ 2.5 GPa). The most compliant of these PEM substrata promoted hepatocyte adhesion and spheroidal morphology. Subsequent modification of PEMs with type I collagen and the proteoglycan decorin did not alter substrata compliance, but enhanced the retention of spheroids on surfaces and stabilized hepatic functions (albumin and urea secretion, CYP450 detoxification activity). Decorin exhibited unique compliance-mediated effects on hepatic functions, down-regulating the hepatocyte phenotype when presented on highly compliant substrata while up-regulating hepatocyte functions when presented on increasingly stiffer substrata. These results show that phenotypic functions of liver models can be modulated by leveraging synthetic polymers to study and optimize the interplay of biochemical and mechanical cues at the cell–material interface. More broadly, these results suggest an enabling approach for the systematic design of functional tissue models applied to drug screening, cell-based therapies and fundamental studies in development, physiology and disease.

Hepatitis B virus (HBV), hepatitis C virus (HCV), autoimmune hepatitis (AIH), and non-alcoholic fatty liver disease (NAFLD) can induce chronic liver disease. The PD-1 inhibitory pathway assists in T cell response regulation during acute and chronic inflammation and participates in the progression of inflammatory liver disease. To examine whether PD-1 and its ligands, B7-H1 and B7-DC, are modulated during chronic necroinflammatory liver disease, we investigated expression profiles in normal patients and patients with the aforementioned conditions. Relative to liver biopsies from normal individuals, those from patients with chronic necroinflammatory liver diseases (HBV, HCV, and AIH) contain increased numbers of PD-1 expressing lymphocytes. Kupffer cells, liver sinusoidal endothelial cells (LSECs), and leukocytes express PD-1 ligands. We also detect PD-1 ligands on hepatocytes within biopsies and on isolated cells. All forms of chronic necroinflammatory liver disease examined correlate with increased B7-H1 and B7-DC expression on Kupffer cells, LSECs, and leukocytes. The degree of necroinflammation correlates with expression levels of PD-1 family members. These results demonstrate that expression of PD-1/PD-1 ligands links more directly with the degree of inflammation than with the underlying etiology of liver damage. The PD-1 pathway may assist the liver in protecting itself from immune-mediated destruction.

Hepatocytes self-assemble in culture to form compacted spherical aggregates, or spheroids, that mimic the structure of the liver by forming tight junctions and bile canalicular channels. Hepatocyte spheroids thus resemble the liver to a great extent. However, liver tissue contains other cell types and has bile ducts and sinusoids formed by endothelial cells. Reproducing 3-D co-culture in vitro could provide a means to develop a more complex tissue-like structure. Stellate cells participate in revascularization after liver injury by excreting between hepatocytes a laminin trail that endothelial cells follow to form sinusoids. In this study we investigated co-culture of rat hepatocytes and a rat hepatic stellate cell line, HSC-T6. HSC-T6, which does not grow in serum-free spheroid medium, was able to grow under co-culture conditions. Using a three-dimensional cell tracking technique, the interactions of HSC-T6 and hepatocyte spheroids were visualized. The two cell types formed heterospheroids in culture, and HSC-T6 cell invasion into hepatocyte spheroids and subsequent retraction was observed. RT-PCR revealed that albumin and cytochrome P450 2B1/2 expression were better maintained in co-culture conditions. These three-dimensional heterospheroids provide an attractive system for in vitro studies of hepatocyte-stellate cell interactions.

The spatial organization of cells of different phenotypes is an important and often defining determinant of tissue function. In tissue engineering, which attempts to rebuild functional tissues from cellular and synthetic components, spatial patterning of cells onto biomaterials is likely to be equally important. We have printed combinatorial arrays of extracellular matrix (ECM) and screened them for attachment by HepG2 hepatocytes, LX-2 hepatic stellate cells, primary portal fibroblasts, and bovine aortic endothelial cells—cells selected as representative phenotypes found in adult liver. Differential cell attachment to the underlying matrix proteins allowed us to establish two-dimensional co-cultures of HepG2 with these non-parenchymal cell types. These general approaches were then translated to tissue engineering scaffolds where deposition of ECM proteins onto electrospun polylactide meshes resulted in patterned HepG2 cultures. We observed that the spatial organization of fibronectin deposits influenced HepG2 attachment and the establishment of co-cultures on our arrays. These micropatterned co-culture systems should serve as valuable tools for studying the soluble and insoluble signals involved in liver development, function, and disease.

Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachmemt and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma cells (HepG2), adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after seven days of culture on only 2 of 18 candidate multilayer systems: (PAH-PSS)n (i.e. n PAH-PSS bilayers) and cross-linked (PLL-ALG)n-PLL. These two systems, as well as cross-linked (PLL-PGA)n-PLL, support attachment and function (in terms of albumin production) of ARH, provided collagen is adsorbed to the top of the film. (PAH-PSS)n, cross-linked (PLL-ALG)n, and cross-linked (PLL-PGA)n-PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.

Cell-based technologies to support/restore liver function represent one of the most promising opportunities in the treatment of acute liver failure. However, the understanding of the constituent cell types that interact to achieve liver-specific structure and function as not been achieved in the development of liver assist devices (LADs). Here we show that hepatocytes migrate toward and adhere and formed sinusoids-like structures in conjunction with liver non-parenchymal cells, and that this liver organoid formed sophisticated tissue after 7 days in an implanted LADs in rodents. Hepatocytes only or in combination with human non-parenchymal liver cell lines (endothelial, cholangiocytes and stellate cells) were cultured in Matrigel, Ultra-structural showed that the hepatocyte-decorated endothelial vascular structures resemble in vivo sinusoids containing plate-like structures, bile canaliculi, and lumen. The sinusoid-like structures retained albumin secretion and drug-metabolism capabilities. In addition, LADs containing cocultures of human liver non-parenchymal cells were transplanted in animals for a week, the liver tissue formed sophisticated structures resembling the liver. These results demonstrate the importance of non-parenchymal cells in the cellular composition of LADs. The novelty of the culture’s sinusoid-like organization and function strongly support the integration of liver non-parenchymal units into hepatocyte-coculture based LADs as a potential destination therapy for liver failure.

Background

Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.

Results

Halichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers.

Conclusion

(I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.